THE BIOSYNTHESIS OF MYCOLIC ACIDS IN MYCOBACTERIUM-TUBERCULOSIS - ENZYMATIC METHYL(ENE) TRANSFER TO ACYL CARRIER PROTEIN-BOUND MEROMYCOLIC ACID IN-VITRO

A closely related family of enzymes from Mycobacterium tuberculosis ha s been shown by heterologous expression to catalyze the modification o f mycolic acids through the addition of a methyl (or methylene) group derived from S-adenosyl-L-methionine (SAM). Overproduction of all six of these enzymes in Escherichia coli and subsequent in vitro reactions with heat-inactivated acceptor fractions derived from Mycobacterium s megmatis in the presence of [methyl-H-3]SAM demonstrated that the imme diate substrate to which methyl group addition occurs was a family of very long-chain fatty acids. Inhibitors of methyl transfer, such as S- adenosyl-L-homocysteine and sinefungin, were shown to inhibit this rea ction but had no effect on whole cells of either M. smegmatis or M. tu berculosis. Purified mycolic acids from M. tuberculosis were pyrolyzed , and the resulting meroaldehyde was oxidized and methylated to produc e full-length methyl meromycolates. These esters were shown to comigra te with a fraction of the acceptor from the in vitro reactions, sugges ting that methyl group addition occurs up to the level of the meromyco late, Protease and other treatments destroyed the activity of the acce ptor fraction, which was also found to be extremely sensitive to basic pH. Antibody to the acyl carrier protein AcpM, which has recently bee n shown to be the carrier of full-length meromycolate produced by a un ique type II fatty acid synthase system, inhibited the cell-free methy l(en)ation of these acids. These results suggest that mycolate modific ation reactions occur parallel with the synthesis of the AcpM-bound me romycolate chain.